All mammals produce steroids which are cleared from the body in varying degrees and excreted through the urine. Different species produce certain chemically unique steroids, while most species produce a number of steroids in common. Most mammalian species produce different relative levels of different steroids at various times during the day, season, and life cycle. Each of these may be excreted in varying degrees and potentially each is recoverable from the urine of such mammal. Steroid content of urine also differs significantly between males and females, between immature and mature members of the species and aged members of the species. Differences also appear between females capable of producing offspring who are pregnant, not pregnant but in estrus, as well as those who are not pregnant and not in estrus.
During the normal life cycle, a woman naturally reaches a point in time where her menstrual cycle stops. This is the basic definition of menopause and is typically characterized by the cessation of ovarian function, leading to a substantial reduction in circulating estrogens. Following the cessation of menses, the decline in endogenous estrogens is typically rapid from 40-250 pg/ml of estradiol and 40-170 pg/ml of estrone during ovulatory cycles to less than 15 pg/ml of estradiol and 30 pg/ml of estrone in postmenopausal women.
Physiologic changes that may result as a women moves from normal menstrual cycles and estrogen levels to an intermediate stage (perimenopause) through menopause to a postmenopausal condition include vulvar and vaginal atrophy causing vaginal dryness, pruritis and dysparenuria, and vasomotor instability manifested by hot flushes. Other menopausal disturbances may include depression, insomnia, and nervousness. Long-term effects of postmenopausal estrogen deprivation may result in significant morbidity and mortality due to the increase in risk factors for cardiovascular disease and osteoporosis. Menopausal changes in blood lipid levels may be precursors to increased incidence of ischemic heart disease and atherosclerosis, and other cardiovascular disease.
Estrogen replacement therapy (ERT) has provided beneficial symptomatic relief for the hot flushes, genital atrophy, symptoms and for the prevention of postmenopausal osteoporosis. In addition, ERT has been shown to be effective in increasing high-density lipoprotein-cholesterol and in reducing low-density lipoprotein-cholesterol giving some protection against cardiovascular heart disease, as well as other beneficial effects. The best known oral ERT available in the United States is a natural mixture of conjugated equine estrogens available under the name PREMARIN®. The Merck Index 13th Edition (2001) defines “conjugated estrogenic hormones” (entry 2533) as “an amorphous preparation containing water-soluble, conjugated forms of mixed estrogens obtained from urine of pregnant mares” and that the principal estrogen present is sodium estrone sulfate. Both Stedman's Medical Dictionary, 26th Edition, p. 600 (1995) and PDR Medical Dictionary, 2nd Ed. p. 622 (2000) define conjugated estrogens in the same manner. The latter two references also define “esterified estrogens” as mixtures of sodium salts of sulfate esters of estrogenic substances. The USP 27 (2004) defines “conjugated estrogens” as a mixture of sodium estrone sulfate and sodium equiline sulfate, derived wholly or in part from equine urine or synthetically from estrone or equiline and contains other conjugated estrogenic substances of the type excreted by pregnant mares. The USP 27 defines “esterified estrogens” as a mixture of the sodium salts of sulfate esters of the estrogenic substances, principally estrone.
For purposes of clarity, the following definitions will be used in the present specification:                1) “esterified steroid” shall mean a steroid that has one or more groups esterified;        2) “sulfated steroid” shall mean a steroid that has one or more sulfate groups esterifying the steroid;        3) “sodium sulfated steroid” shall mean the sodium salt of the sulfate ester of the steroid;        4) “esterified estrogen” shall have the meaning given in the USP 27 set forth above;        5) “conjugated estrogens” shall have the meaning given in the USP 27 set forth above.Unless the specific context of the text requires otherwise, the use of the term “salt” in conjunction with either of 1) or 2) above shall mean a salt selected from the group consisting of alkali metal salts, alkaline earth metal salts, and ammonium salts, with particular preference for the alkali metal salts, and special preference for the sodium salt.        
Unfortunately, elevated estrogen levels have been related with an increased risk of endometrial cancer. Thus, the minimal dose of estrogens to achieve the desired result is essential to adequately balance the risks and benefits of estrogen therapy. The addition of progestin to therapy also lessens the endometrial cancer risk and for many women combination hormone replacement therapy (HRT) is a rational approach.
As noted, natural mixtures of conjugated estrogens (such as are found in the urine of pregnant mares) have proven particularly effective and well tolerated for ERT and HRT. The dissolved solids content in the urine of pregnant mares (PMU) may naturally vary within wide ranges, and generally lie in a range of about 40-90 g of dry matter per liter of urine. In addition to urea and other usual urine contents PMU generally contains about 2 to about 5% (of the dry matter—i.e. about 0.8 g/l to about 4.5 g/l) of phenolic constituents, which include cresols and dihydro-3,4-bis[(3-hydroxyphenyl)methyl]-2(3H)-furanone (HPMF), which may be present in free form or as salts of the sulfate esters thereof. Furthermore, the PMU has a natural mixture of estrogens that are largely present in conjugated form (i.e. as the sulfuric acid semi-ester sodium salt, also referred to as the “sulfate salt”). These sodium sulfated estrogens are typically present in an amount of about 0.3% to about 1% relative to the PMU dry matter (i.e., about 0.12 g/l to about 0.9 g/l of PMU).
In the early days of conjugated estrogen extraction from PMU, liquid-liquid extractions were employed using organic solvents that were immiscible or only slightly miscible with water, such as ethyl acetate, n-butanol, isobutanol, or cyclohexanol. However, such liquid-liquid extraction procedures resulted in problems such as severe foaming, sedimentation, emulsification, and poor phase separation, and several extraction steps were usually required. This led to processing losses and only partial recovery of the estrogen content of the PMU. Liquid-liquid extraction processes are disclosed in, for example, U.S. Pat. Nos. 2,551,205; 2,696,265; 2,711,988; 2,834,712 and WO01/27134 (corresponding to PCT/US99/23730) (all of which are incorporated herein in their entirety by reference).
Solid extractions using resins were first proposed by Bradlow (Steroids 11:265-272 (1968)) wherein a neutral, non-polar hydrophobic polystyrene resin, Amberlite XAD-2 (Rohm and Haas), was used. According to Bradlow, optionally diluted urine is passed through an Amberlite XAD-2 packed column using a low rate of flow, which absorbs the conjugated estrogens (although the absorption capacity is low), and the absorbed estrogens are eluted with methanol or ethanol. While this my have been suitable for an analytical process, the low rate of absorption did not make it practical as a commercial extraction process for obtaining large quantities of the steroids.
Other solid extractions of conjugated estrogens from PMU have been described in U.S. Pat. No. 3,769,401 (using a polyamine anion exchange resin (such as Dowex 1-X-2)); U.S. Pat. No. 5,723,454 (using a semi-polar porous, non-ionic adsorber resin (such as a cross-linked aliphatic polycarboxylic acid ester resin for example Amberlite XAD-7); U.S. Pat. No. 5,814,624 (using a hydrophobized silica gel a.k.a. a reverse-phase silica gel); US 2002/0156303 (using a polystyrene divinylbenzene resin); US 2003/0105344 (using a resin to extract all estrogenic components of the natural mixture in the PMU so that the volume to be transmitted to the laboratory from the field is reduced); and US 2003/0215953 using silicone membranes to separate the cresol components from the PMU before contacting the cresol reduced PMU with an adsorber resin; all of which are incorporated herein by reference. In each case, the adsorbed conjugated estrogens are eluted from the resin using a water-miscible organic solvent. Each of the foregoing US patents and applications is incorporated in its entirety herein by reference.
In addition to the processing issues themselves, the estrogenic content of PMU varies from batch to batch, even with respect to the currently marketed product PREMARIN. The total estrogen content varies as well as the presence or absence of some of the estrogenic components depending upon the mare, and what stage of pregnancy the mare is in when the urine is collected. Thus, the simple extraction of “estrogens” or of “conjugated estrogens” leads to a “conjugated estrogen product” that varies both in relative concentration (one steroid component relative to another steroid component) as well as in the presence of a number of the “estrogenic species” (some species may be missing from one batch and present in another). This has led to difficulties in development of a generic equivalent of PREMARIN, mostly because of the lack of a defined reference standard and the complex nature of the product.
Despite the advances in the extraction of estrogenic materials from urine, the processes can be improved upon in terms of yields, volumes of materials needed for processing the urine; and especially in consistency of product that results from the process. In addition, given the size of the market for conjugated estrogens, a source of a generic alternative has and continues to be desired.
In addition, during the extraction process for obtaining a particular set of steroids, many other steroids of commercial value are obtained and can be isolated with relatively small additional cost, or can be obtained as commercially valuable mixtures of different sets of steroids useful for human, veterinary, agricultural, and chemical purposes.